The purpose of the proposed study is to dissect the cellular vectors of adoptive immunotherapy utilizing tumor-infiltrating lymphocytes (TIL), in order to achieve insight into the nature and mechanisms of TIL-mediated tumor eradication. TIL will be studied in a murine model system utilizing methylcholanthrene-induced MCA105 sarcoma, the colon adenocarcinoma MC-38 and the melanoma B16. TIL,s are now generated from each of these tumors and expanded in IL-2 and used in adoptive transfer experiments. Study of the optimal conditions for growth of TIL in vitro and their effectiveness in vivo will be undertaken. TIL grown from each tumor will serve as specificity control for the other tumors. Clones will be generated by treating bulk populations with monoclonal antibody and complement and these clones or subpopulations will be compared to bulk TIL cultures and LAK cells for in vitro growth characteristics and in vivo efficacy. Activation of murine TIL's with anti-CD3 (2C-ll) will be studied in vitro and in vivo utilizing a subdermal tumor model and sequential sacrifice of mice during tumor rejection will be performed. Samples of tumor will be studied immunohistologically. These studies will document the cell populations involved in tumor rejection. The role of cytoxan in these adoptive immunotherapy models will be studied, specifically to separate the cytoreductive and immunosuppressive effect of this drug, and optimal dose and schedule will be determined. Parallel experiments will be performed utilizing human TIL's. Optimal conditions for growth of these cells will be defined. The role of anti-T3 and other antibodies such as WT31 which induce activation of human TIL's will be studied. The phenotype and function of TIL bulk cultures will be studied, paying particular attention to the T4/4B4 population as defined by 2-color immunofluorescence in conjunction with cell sorting, to isolate T4/4B4 positive T cells from TIL bulk culture. TIL phenotypic changes over time, and characterization of optimal conditions for growth and tumor specific cytotoxicity will be defined. Clones will be generated from human TIL's and studied in vitro for growth and function. At the completion of this grant, there should be a much better understanding of the underlying mechanisms and nuances of TIL in the mouse and human which will be applied toward the design of new immunotherapy trials in the human.